The Problem: We know that parental asthma is a major risk factor for asthma in children (maternal > paternal), and that exposures to chemicals and pollutants during pregnancy can increase risk of future disease. In contrast, it remains unknown whether pregnancy exposures of the developing fetus can cause true transgenerational effects---i.e. increased asthma susceptibility in the F3 offspring (progeny of the F1 fetus and its F2 ova). The Solution: Our laboratory has already established a robust model of maternal transmission of asthma susceptibility. A single exposure of normal pregnant mice to diesel exhaust particles (DEP) causes enhanced allergic responses and an asthma-like phenotype in F1 offspring, linked to epigenetic changes in offspring dendritic cells (DCs.) this groundwork provides a robust platform to test whether transgenerational inheritance of asthma susceptibility will follow pregnancy-exposure to a prototypical environmental toxin. Aim 1 will investigate transgenerational transmission of asthma susceptibility to F2 and F3 offspring after a single pregnancy exposure to diesel exhaust particles (DEP) of F0 mother mice. For all 3 generations, we will measure allergic airway inflammation (BAL eosinophils, cytokines, histology) and airway hyperresponsiveness (AHR) after an intentionally sub-optimal allergen (ovalbumin) sensitization and aerosol challenge protocol that has minimal effects in normal pups. Dose and timing studies will follow initial trials using parameters already shown to have F1 effects. Aim 2 will map DNA methylome changes in 'susceptible' DCs from F1, F2 and F3 generations. This aim builds on prior work showing a distinct methylome in F1 DCs of asthma-susceptible pups born to DEP-exposed mothers. We will track the methylome changes that accompany persistence (or disappearance) of asthma susceptibility from F1 to F2 and F3 progeny of DEP-exposed mother mice. To do this, we will use reduced-representation bisulfite sequencing (RRBS) to quantify and map DNA methylation changes in splenic DCs, with additional validation of selected targets by pyrosequencing. Bioinformatic analysis will identify patterns and magnitude of methylation changes seen in every generation, and allow direct correlation to the phenotype observed in littermates (susceptibility and magnitude of allergic inflammation, AHR). Significance: The project will determine if transgenerational inheritance of asthma susceptibility occurs, and lays the foundation for better mechanistic analysis of persistence or resolution of pregnancy exposure effects.